Novel two-photon caged GABA compounds Understanding how GABAergic inhibition works is necessary to decipher the function of brain and the pathophysiological processes of diseases that affect it, including many epilepsy and addition syndromes. At the same time, there is no unified agreement as to how exactly GABAergic inputs function and even the basic question of whether they are inhibitory or excitatory is actively debated. Part of the reason for these controversies is the fact that inhibitry inputs target subregions of the postsynaptic cells, and there are not good tools to investigate these inputs with high spatial resolution. Local activation of receptors in living neurons can be achieved by two-photon photorelease of caged compounds. Indeed, two-photon uncaging of glutamate has revolutionized current understanding of excitatory transmission and integration in mammalian neurons. Unfortunately, opto-chemical tools to photorelease GABA are scant, even though they would be extremely useful to study the function of GABAergic inhibition. In this proposal we introduce a novel family of two-photon caged GABA compounds. Specifically, we will test and characterize the two-photon release of three different chemical generations of caged GABAs and use the best ones to generate high-resolution maps of GABAergic responses on living pyramidal neurons from mouse neocortical slices. Finally, we will confirm the accuracy of these maps using a novel 3D high- throughput electron microscope to identify symmetric synapses. The proposed work will expand the chemical toolbox of biological uncaging to include novel high-quality caged GABA compounds that can be photo-released with two-photon lasers. These new compounds will enable the detailed investigation of the functional effects of GABAergic transmission on selective subcellular compartments, something likely to have a major impact on our understanding of how inhibition alters normal and diseased brain function, since some of these compounds can be used to control epilepsy. Finally, our data will reveal, for the first time, the functional effect of GABAergic inputs onto dendritic spines. Since spines mediate most excitatory connections, these results could also alter our understanding of how excitatory inputs are integrated. PUBLIC HEALTH RELEVANCE: Novel two-photon caged GABA compounds Although GABAergic circuits mediate most of the inhibition in the brain, and are affected in many brain pathologies, their function is poorly understood, partly because they act with great spatial selectivity onto subregions of the neurons. We propose the development of novel opto-chemical tools that will allow to optically activate GABAergic inputs onto neurons with unprecedented spatial resolution, thus enabling the dissection of their functional properties. Our work could result in the generation of novel optical methods to control the activity of neurons in hyper-excitable pathological states such as in epilepsy. !